The present invention relates to a device for measuring fluorescence or phosphorescence emitted from a sample irradiated with excitation light, and more particularly to a fluorescence and phosphorescence measuring device capable of obtaining high detection sensitivity and SN ratio.
A fluorescence and phosphorescence measuring device detects a target component in an article to be measured using a phenomenon that when excitation light such as an excitation lamp or laser light is irradiated to a sample to be measured, the sample emits light with a wavelength different from the wavelength of the irradiated excitation light.
In an actual measuring device, at the time of measuring target emitted light from a sample, due to irradiation of exciting light to a sample container and an existing lens and filter, emitted light, fluorescence, or phosphorescence other than light from the target is measured and the measured SN ratio may be reduced.
Further, when light is measured on the confronting side of irradiating light, the afterglow of exciting light enters into a light measuring device and the SN ratio may be reduced in the same way. Namely, fluorescence or phosphorescence (hereinafter referred to as measurement interference light) not from a sample is analyzed and measured as if the article to be measured is included in the sample though the article to be measured is not naturally included in the sample and an incorrect judgment may be made.
Further, scattered light or measurement interference light is varied in intensity, as a result, the minimum detection limit of an applied analytical measuring method or a measuring device is remarkably reduced.
Conventionally, in order to reduce scattered light resulted from a lamp, a laser, an existing lens and filter, or a sample container, or interference light disturbing measurement, fluorescence is analyzed beforehand, and a raw material having smaller measurement interference light is selected and used as a material, and the use amount of material is reduced by making the material thinner, or the material surface is coated.
Further voltage, shape, and gas pressure were studied and the lamp and laser were improved so as to make an afterglow as little as possible after end of irradiation. With respect to the light measuring method, in addition to the transmission surface fluorescence measuring method that the exciting light irradiation direction coincides with the light measuring direction, the side fluorescence measuring method that the direction does not coincides and light from the lamp and laser is not directly taken in the detection unit and the irradiation surface fluorescence measuring method are properly used.
Furthermore, by development of a phosphor and a luminous body, the difference between the exciting light wavelength and the fluorescence or phosphorescence wavelength, the so-called stokes shift is increased, and a wavelength selection filter is installed, and avoidance of detection of light with a wavelength adjacent to the exciting light wavelength is executed.
Further, the time-resolved measuring method using a luminous body having a long fluorescence emission and phosphorescence emission continuous time and measuring fluorescence or phosphorescence during a time different from the exciting light irradiation time has been developed. In recent years, development of a luminous body particularly using a rare-earth element (Lantanoide) such as europium having a long stoke shift and a long light emission continuous time has been progressed and a method for labeling an article to be measured with it and executing analytical measurement has been improved.
However, in every method, scattered light or interference light disturbing measurement cannot be reduced sufficiently and an occurrence of interference light from articles other than a sample cannot be avoided.
In the time-resolved fluorescence measuring method, exciting light is said not to be taken in the light measuring device, and therefore, it is a normal way to set the fluorescence measuring time to the time that the exciting light is off. For example, it is a general method to turn on a flash lamp at 1000 Hz for 0.01 ms, measure between 0.4 ms and 0.8 ms after the lamp turns on, measure light in a 1-ms cycle, calculate the light quantity for 1 second, and display the data.
In a fluorescence measuring device or a phosphorescence measuring device, as a light source, a tungsten lamp, a mercury lamp, an argon gas lamp, a xenon flash lamp, a nitrogen gas laser, or an argon gas laser is used. As a method for preventing light other than the target light from irradiating a sample, a light shielding plate or an optical path shut-off plate (hereinafter referred to as a chopper) may be used.
On the circumference having a circular black plate, a gap with a fixed size is formed, and the black plate is rotated, and exciting light generated from the light source passes only through the gap, thus exciting light at a fixed time interval is irradiated to a sample.
In the light source, when the lamp is turned off, the light does not disappear instantaneously and the core of the light source reduces the light for a little while, thus even if the voltage is cut off, the light source generally emits light. The afterglow after the voltage is cut off lets the lens, filter, sample, or sample container of the light source or detection unit emit light sometimes, causing increasing of noise or background at the time of measurement.
Furthermore, the afterglow has a wavelength different from the original one of the light emitted from the light source, so that when exciting light is to be separated from fluorescence or phosphorescence in wavelength, the afterglow may have the same or close wavelength to that of fluorescence or phosphorescence, causing increasing of noise at the time of measurement. In order to remove the afterglow of the light source, it may be set so that the chopper ends breaking at the same time with or prior to turning off the power source of the light source.
In the time-resolved fluorescence measuring method, the exciting irradiation time to a sample and the measuring time of fluorescence or phosphorescence emitted from the sample are made different from each other, thus measurement of noise resulted from the exciting light is reduced and the sensitivity of measurement is increased.
In order to reduce the aforementioned noise or background, the chopper is put in front of the sample or the lens and filter of the light source and light other than the target is prevented from irradiation to the sample. Breaking by the chopper is synchronized with turning on or off the power source.
Furthermore, the lens, filter, or mirror receiving irradiation of light emitted from the light source may scatter the light. Scattered light is preferably prevented from irradiation to the sample. In order to prevent the afterglow of the lamp after the power is turned off from irradiation to the sample, the chopper is put on the optical path connecting the lamp and sample and light other than necessary light physically emitted from the light source is shielded.
By doing this, when the lamp is turned off, unnecessary light is prevented from irradiation to the sample and the sample emits fluorescence or phosphorescence, only when it obtains the target energy.
In the same way, a physical shutter is installed or an electrical circuit shutter is set just behind the sample, thus exciting light is prevented from irradiation to the light quantity detector like photomultiplier tube. By doing this, noise resulted from exciting light can be reduced and afterglow of the light source emitted after irradiation of exciting light and afterglow or scattered light from the lens, filter, mirror, or sample holder can be removed.
As a time-resolved fluorescence detection method using such a chopper, the art disclosed in Japanese Laid-open Patent Publication No. Hei 10-267844 is known. The art uses a first chopper so as to convert light to be entered to a sample to pulse light using the light source of exciting light as a continuous light source and uses a second chopper so as to detect only the target fluorescence or phosphorescence among light transmitted from the sample. By this method, afterglow of the light source and afterglow or scattered light from the lens, filter, mirror, or sample holder can be removed efficiently.